Method of separating whole blood



United States Patent 3,464,890 METHOD OF SEPARATING WHOLE BLOOD TheodoreE. Weichselbaum, St. Louis, Mo., assignor, by mesne assignments, toBrunswick Corporation, Chicago, 111., a corporation of Delaware NoDravw'ng. Filed Mar. 1, 1965, Ser. No. 436,300 Int. Cl. C12d 13/02; C12k1/10 US. Cl. 195-66 2 Claims ABSTRACT OF THE DISCLOSURE A solid granularmaterial, e.g. polystyrene beads, having a coating of anti-coagulant,e.g., heparin, and having a specific gravity intermediate that of plasmaand blood which can be used by adding to blood in vitro to separateplasma from the remainder of the blood. When the beads are added towhole blood, a separation occurs by which the plasma portion of theblood forms, by virtue of the presence of the anti-coagulant, in theresulting layer above the beads while the remaining portions of theblood form in a layer below the beads so that the plasma can easily beseparated. In a preferred form, the material used to coat the beads alsoincludes a wetting agent.

This invention relates to the separation of whole blood into plasma andred cell components. More particularly, this invention relates to suchseparation of blood wherein coagulation of blood by the blood clottingmechanisms is stopped so that whole plasma is recovered which containsan amount of prothrombin and/ or clotting agents, in the same ratio assuch clotting agents are present in the total plasma portion of thewhole blood. The term whole plasma, as used herein, refers to plasmawhich contains clotting agents in an amount proportional to the ratio ofclotting agents:plasrna in the whole blood, and the present inventionassures separation of such plasma without clotting of the blood.

It has recently become a common technique to separate serum from othercomponents of whole blood by using a particulate material, e.g.polystyrene particles or pellets, having a specific gravity intermediatethe specific gravity of blood serum or plasma and the red blood cells.Accordingly, a sample of blood is permitted to clot in vitro. A quantityof the particulate \material is introduced into the sample and themixture is stratified by centrifuging or the like. The serum forms a toplayer above the particulate material, and the blood cells form a bottomlayer below the particulate material. The particulate material is usedin an amount sufficient to form a tight layer between the two separatecomponents. The serum may then be readily recovered as the top la er.

Ihe above procedure is effective in obtaining serum from whole blood.However, where it is desired to obtain plasma, which differs from serumby the presence of clotting agents, the procedure is not sufiicientlyprecise to assure that the clotting agentstplasma ratio of the separatedplasma is the same as the clotting agentzplasma ratio of the wholeblood. Assuring such ratio is important for a number of reasons. Forexample, it is often necessary to estimate the prothrombin content ofthe blood sample from a patient in order to determine and controldosages of anti-coagulant drugs, such dicumerol (bis-hydroxycoumarin).In such cases it is, of course, necessary to assure that the plasma isreally whole plas- The above technique for obtaining serum has alsopreviously been used in attempts to obtain plasma, except that ananti-coagulant is added to the blood sample and the blood is notpenmitted to stand. However, some clotting still takes place, therebyupsetting the ratio of clotting agents in the plasma recovered.

It is a general object of this invention to produce or separate wholeplasma from blood and particularly from the red blood cells, in a newand improved manner assuring an absence of blood coagulation.

It is further an object of this invention to recover Whole plasma fromwhole blood, wherein the whole plasma contains the same proportion ofanti-clot agents to plasma as is present in the whole blood.

Another object of this invention is to provide a new and usefulcomposition which can be used in combination with polystyrene particlesor other particulate material having a specific gravity intermediatethat of the red cells and plasma for assuring separation of wholeplasma.

Yet another object of this invention is to provide, as a novelstructure, a particulate material having a specific gravity intermediatethat of plasma and red cells and having a dried coating thereoncontaining an anti-coagulant, which structure is useful in separatingwhole plasma in accord with any of the foregoing objects.

A still further object of this invention is to provide a new and usefulblood component separation system and method in which an anti-coagulantcan be carried throughout a blood sample in vitro by a solid particulatematerial having a specific gravity intermediate that of the plasma andred cells and having the anti-coagulant carried as a coating on theparticulate material.

Other objects may be apparent to those in the art from the disclosureherein.

It has now been found that the whole plasma component of whole blood canbe precisely recovered from whole blood in vitro to provide a plasmacomponent which contains the same ratio of clotting agentszplasma orclotting agentstserum as was present in the whole blood startingmaterial. The present invention provides particulate material having aspecific gravity intermediate that of the plasma and red cells, and thesurface of the particulate material is coated with and carries ananticoagulant. In the most advantageous form, the particulate materialsurface is wetted with a water soluble non-ionic wetting agent toincrease its capacity for absorption and carrying of the anti-coagulant.Preferably, the anti-coagulant is present on the particulate materialstructure in a known amount based on the weight of the particulatematerial.

The particulate material can be any material which is insoluble in anaqueous medium and which is non-reactive or inert with respect to theblood, wetting agent or anticoagulant. For example, the polystyreneparticles or pellets used in the separation of serum from Whole bloodcan be used herein. Other suitable and usable plastic, and evennon-plastic, materials which have the proper specific gravityintermediate that of the plasma and red cells will be evident. Where thematerial has a good surface absorption capacity, the wetting agent mayprove wholly unnecessary. However, it is preferred to use the wettingagent with plastic particles since such particles are often not capableof absorbing and holding the anti-coagulant on their surfaces in theabsence of the wetting agent.

In forming the coated particles of this invention, the particulatematerial or particles are coated with the anticoagulant, e.g. inadmixture with a wetting agent or after coating of the particles with awetting agent, and the coated structure is then dried to provide a drycoating. The amount or concentration of wetting agent and amount orconcentration of anti-coagulant used during the coating step will dependto some extent on the surface area and absorption characteristics of theparticle being coated,

as is well known in the absorption of liquids on solid surfaces. For aparticular size and nature of solid particle, the amount absorbed orabsorbable can be readily determined by simple experimentation, i.e. byperforming a test run of the coating method to determine how much liquidis actually absorbed per unit of particulate material.

The present method may advantageously be carried out by supporting theparticles on a foraminous structure such as a screen and pouring thewetting agent and anti coagulant solution or solutions over theparticles. Preferably, in runs standardizing the method, the screenshould be pre-treated with additional coating materials so that theamount absorbed by the screen does not affect the calculation of theamount absorbed by the particles and the amount, e.g. by weight, ofparticulate material used should be predetermined. Where the amount andconcentration of solution poured over the particles is known and thesolution draining from the screen is collected, the amount absorbed canreadily be determined. Since the amount absorbed by the particles isknown, the amount per unit weight of particles can be readilycalculated. The particles are dried, e.g. by placing in a vacuum oven,after coating, so that the anti-coagulant does not flow from theparticle surface. The particles can then be packaged and labeled withtheir concentration. If necessary or desired, prior to packaging, theparticles from each run can be diluted with uncoated particles orparticles carrying lesser amounts of anti-coagulant or can beconcentrated with particles carrying greater amounts of anticoagulant toprovide a standardized mixture of particles in which the amount ofanti-coagulant per unit weight of particle is precisely known.

The water soluble non-ionic wetting agents are preferably used in anaqueous solution containing a small amount, e.g. from .02 up to 20% andpreferably .2 to 2% of the wetting agent. The wetting agents often cancause rupture of red blood cells, in whole blood or otherwise adverselyaffect the red blood cells, and therefore it is preferred that theamount of wetting agent be suflicient to wet the surface of theparticulate material with only litle excess, if any. Thus, where thesurface of the particulate material is more highly absorptive or has agreater surface area, weaker solutions of wetting agent will be usedwhile the stronger solutions will be used where surface area orabsorptivity is low.

The anti-coagulant is used in a concentration sufficient to provide theproper amount of anti-coagulant on the particulate material surface forpreventing coagulation of the blood. Thus, the amount of anti-coagulantper gram of particulate material will depend upon the amount of blood towhich a given weight of particulate material is intended to be added.Anti-coagulant materials such as heparin are very expensive, so for sakeof economy it is preferred that they not be used in excess above theamount necessary, although no excess appears to adversely affect theblood. Proper amounts of anti-coagulant for addition to blood are wellknown and sufficient amounts of anticoagulant should be used to coat theparticles to provide the proper amount in the blood during separation ofplasma. The amount of anti-coagulant is usually sufiicient to provideabout 10 U.S.P. units of heparin per ml. of blood, although lesseramounts, e.g. down to 1 U.S.P. unit, can be used where lesser amounts ofanti-coagulant activity are desired. Of course, excesses up to 100U.S.P. units and higher can be used, but at a cost disadvantage. Usuallyfrom about one to about 1000 U.S.P. units of coagulant per gram ofparticulate material will be sufficient for any application.

Anti-coagulants are obtainable in prepared solutions, or concentrates ofanti-coagulant can be diluted with water to prepare a solution of aknown strength, e.g. usually in the range of l2000 U.S.P. units per ml.Stronger or weaker solutions can be prepared, and it may be desirable todo so in the present invention, especially with respect to strongersolutions, since many of the more 4 readily available usable particulatematerials have relatively low surface areas per unit of weight, due to alarge particle size.

The following specific examples are given in illustration of the presentinvention and are not intended to be limiting on the invention.

Example I 500 grams of polystyrene pellets having an average diameter ofV inch and an average length of A inch are supported on a retainingscreen which has been previously dipped in a 2% solution of liquid watersoluble polyethylene glycol (averaging tetraethylene glycol) andthereafter dipped in a heparin solution containing 1000 U.S.P. units ofheparin per ml. and dried. 250 mls. of the polyethylene glycol solutionare mixed with 250 mls. 0f the heparin solution, and the resultingmixture is then poured over the polystyrene pellets. About of thecombined solution is recovered beneath the screen, indicating absorptionof about 20%, or about mls., by the pellets. The coated pellets are thenquickly dried in a vacuum oven until the Water has been evaporatedtherefrom, leaving a dry coating of the anti-coagulant.

Example 11 The screen is prepared as above, and 250 mls. of thepolyethylene glycol solution are poured over a fresh 500 grams ofpolystyrene pellets supported by the screen. The coated pellets are thenvacuum oven-dried until water is evaporated having a waterless or drycoating of the polyethylene glycol. 250 mls. of the heparin solution arethen poured over the pellets and the coated pellets are again dried byevaporation of Water in the vacuum oven. The total amount of solutioncollected indicated that approximately the same amount of wetting agentand heparin was absorbed on the surfaces of the pellets.

In the above two examples, pellets were prepared having dried coatingscontaining 100 U.S.P. units of heparin per gram of pellets.

Example III An anti-coagulant solution of 10% neutral sodium oxalatesalt is prepared. 2% polyethylene glycol is added to the solution, andthe procedure of Example I is repeated using the resulting solution. Theresulting coated polystyrene pellet product has a dry coating whichcontains approximately 200 U.S.P. units of sodium oxalate per gram ofmaterial.

Example 1V 2 grams of pellets prepared as in Example I with the drycoating containing 100 U.S.P. units of heparin per gram were added to 10mls. of fresh Whole blood in a test tube. The test tube was closed andshaken vigorously for a few seconds. The tube was then spun in acentrifuge until the red cells had settled to the bottom of the tube.The materials in the tube appeared in three strata, bottom red cellstratum, an intermediate stratum of the pellets and an upper stratum ofplasma. The anti-coagulant had been desorbed from the pellets. The upperplasma layer was poured off from above the layer of pellets, the pelletsforming a firmly packed layer and retaining the red blood cells withinthe test tube. When compared with an analysis of the blood, it was foundthat the plasma contained the same ratio of clotting agents, i.e.prothrombin and fibrinogen, per unit of plasma, as was present in theoriginal whole blood per unit of plasma.

Although heparin and sodium oxalate have been used in the above examplesas anti-coagulant materials, it is to be undertsood that any watersoluble salt of oxalic acid or citric acid, including, but not limitedto, the sodium, potassium and ammonium salts, may be used in lieuthereof. Sodium or other citrate or oxalate functions as ananti-coagulant by preventing the prothrombin from being converted tothrombin. Other useful anti-coagulants include hirudin and thechondroitin sulfuric acid anti-coagulants which contain chondroitinsulfuric acids or esters thereof as active ingredients thereof. Amongthe latter anti-coagulants is heparin, which functions as ananticoagulant by blocking the reaction of "thrombin with fibrinogen, areaction which would otherwise form fibrin to clot the blood.

The water soluble non-ionic wetting agents are well known, and any suchwetting agents can be used. Particularly preferred are the water solublepolyhydroxy hydrocarbon and polyhydroxy others such as alkylene andpolyalkylene glycols, e.g. ethylene glycol, propylene glycol, diethyleneglycol, triethylene glycol, tetraethylene glycol, and other polyethyleneglycols and polypropylene glycols, pentaerythritol, etc. Other suchwetting agents include, but are not limited to water soluble alcoholssuch as methanol, ethanol and propanol, amides such as formamide andbutyramide, ketones and aldehydes such as acetone, methylethylketone,acetaldehyde and butyraldehyde, ethers such as ethyl ether andmethylethyl ether, and other water soluble organic compounds having atleast one non-ionic polar group, and mixtures of such compounds. Wherecompounds herein are described as water soluble, it is intended 'thatthe compounds be soluble in the amounts and under the conditions used.

The coated particles can be supplied in a glass container approximatelyof test tube dimensions such as the well known and available taperedbottom test tubes conventionally used in gravitational separations ofblood samples. The container can have a sealed or stoppered top forpreventing contamination. When it is desired to conduct a bloodseparation, the seal or stopper can be removed and the blood can beintroduced and agitated in the presence of the coated particles and themixture can be permitted or caused to separate according to specificgravities. Thus, although the present invention is described withrespect to addition of the coated particles to the blood, it is fullyintended that such addition includes the introduction of blood into acontainer which already contains the coated particles.

The foregoing detailed description is given for clear- 6 ness ofunderstanding only and no unnecessary limitations are to be understoodtherefrom, as some modifications will be obvious to those skilled in theart.

I claim:

1. The method of separating plasma from whole blood which comprisesbringing into contact with the blood a separating amount of inertparticulate material having a specific gravity intermediate that ofplasma and red cells and having a dry coating of an anti-coagulantamount of anti-coagulant on the surface thereof in an amount sufiicientto form a barrier layer between the plasma and red cells, agitating theparticles and whole blood, centrifuging the resulting mixture andrecovering the plasma from above the particulate material.

2. The method of separating plasma from whole blood, which methodcomprises bringing into contact with the blood a separating amount ofinert particulate material having a specific gravity intermediate thatof plasma and red cells, said particulate material having a dry coatingcomprising a surface wetting amount of wetting agent and ananti-coagulant amount of an anti-coagulant, agitating the resultingmixture, centrifuging the resulting mixture, and recovering thestratified layer above said particulate material as whole plasmacontaining the blood clotting agents in an amount proportional to theratio of clotting agents to the whole plasma in the whole blood.

References Cited UNITED STATES PATENTS 3,094,466 6/1963 Schwartz -l03.53,234,096 2/1966 Pollack 167-845 3,268,401 8/1966 Birnbaum et al. 16738OTHER REFERENCES A textbook of Clinical Pathology, Miller, Sixth cd.,chapt. l, L. W. Diggs, M.D., pp. l-3.

FRANK CACCIAPAGLIA, 111., Primary Examiner

